iotechnology of
`Antibiotics
`
`Second Edition, Revised and Expanded
`
`edited by
`William R. Strohl
`The Ohio State University
`Columbus, Ohio
`
`MARCEL
`
`DEKKER
`
`MARCEL DEKKER, INC.
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`NEw YORK' BASEL' HONG KONG
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`EXHIBIT NO. 1008 Page 1 of 23
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`ISBN: 0,8247,9867,8
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`EXHIBIT NO. 1008 Page 2 of 23
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`

`Lipopeptide Antibiotics Produced by
`Streptomyces roseosporus and
`Streptomyces fradiae
`
`Richard H. Baltz
`Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana
`
`I.
`
`INTRODUCTION
`
`Streptomyces roseosporus NRRL11379 produces A21978C, a complex of acidic lipopeptide
`antibiotics (1). The cyclic depsipeptide portion (Figure 1a) contains 13 amino acids
`cyclized to form a lO-amino-acid ring. The A21978C factors contain different 10-, 11-,
`12-, or 13-carbon fatty acids attached to the amino-group of the terminal L-Trp (1). The
`fatty acid side chains are readily removed by incubation with Actinoplanes utahensis (2),
`and the cyclic peptide can be reacylated at the amino-terminus of Trp to produce semi(cid:173)
`synthetic acyl, aroyl, and extended peptide derivatives (3). The n-decanoyl analog of
`A21978C, LY146032 or daptomycin, is a potent antibiotic active against Gram-positive
`bacteria, including methicillin-resistant Staphylococcus aureus, methicillin-resistant
`Staphylococcus epidermidis, vancomycin-resistant enterococci, and penicillin-resistant
`Streptococcus pneumoniae (see Section IV). Streptomyces fradiae A54145 also produces a
`complex of cyclic lipopeptides containing 13 amino acids with a 10-amino-acid ring
`(4,5). The A54145 complex is similar to daptomycin in that different factors contain
`various long-chain fatty acids attached to the N-terminal Trp (Figure 1b). A54145 differs
`from daptomycin in that the cyclic peptide is variable in positions 12 and 13, and all four
`possible peptides are observed in normal fermentations (4,5).
`In addition to the N-terminal Trp, A54145 contains some amino acids identical or
`similar to those in daptomycin in other positions, including the position 4 Thr, which
`participates in the ester linkage that closes the ring. A54145 factors have in vitro antibac(cid:173)
`terial properties similar to those of daptomycin (see Section IV). The fatty acyl side
`chains of A54145 can be removed by incubation with A. utahensis (6), and the N-termi(cid:173)
`nal Trp can be reacylated (6), just as with the daptomycin nucleus (2,3).
`The similarities in the structures of these two lipopeptides suggest that the biosyn(cid:173)
`thetic pathways may have evolved from a common ancestral pathway. Thus, the study of
`the structural organization and physical map locations of the biosynthetic gem:s may pro(cid:173)
`vide insights into the evolution of these complex lipopeptide pathways. Further, the
`diversity in amino acid sequence in these related molecules suggests that further mod in-
`
`415
`
`PETITIONERS
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`

`416 Baltz
`
`cation of the peptide portion of these cyclic peptides may generate as yet unknown, but
`related antibiotics-some of which may prove to be superior to either of the parent mol(cid:173)
`ecules. In this chapter, I summarize what is known about the genetics, biosynthesis, mode
`of action, and antibacterial activity of these antibiotics. I also speculate on how the activ(cid:173)
`ities of these molecules may be further altered by modifying the genes encoding the mul(cid:173)
`tienzyme peptide synthetases involved in determining the amino acid sequences of the
`peptide portion of the molecules.
`
`II. BIOSYNTHESIS Of LlPOPEPTIDES
`A. Daptomycin Biosynthesis
`S. roseosporus produces A21978C, a complex of lipopeptide antibiotics highly active
`against Gram-positive bacteria (1). The A21978C complex was separated into three
`major factors, Cl' Cz' and C3, and three minor factors, C4, Cs' and Co (Table 1). All six
`factors contain an identical 13-amino-acid core cyclic peptide, containing 11 common L(cid:173)
`or D-amino acids and 2 unusual amino acids, 3-methyl glutamic acid (3mGlu), and the L(cid:173)
`tryptophan metabolite L-kynurenine (L-Kyn). Enzymatic studies employing glutamine
`synthetase and L-glutamic acid decarboxylase established that the stereochemistry of the
`glutamate analog as L-threo-3-methyl glutamic acid (1). The linear sequence of the pep(cid:173)
`tide core was established as L-Trp-L-Asn-L-Arg-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L(cid:173)
`Asp-Gly-D-Ser-L-3mGlu-L-Kyn. The cyclic depsipeptide is formed by an ester linkage
`between L-Kyn and the L-Thr hydroxyl-group (Figure 1). The factors Cl' Cz' and C3 were
`shown to contain anteiso-undecanoyl (Cll), iso-dodecanoyl (C12), and anteiso-tride-
`canoyl (C13) side chains, respectively. The minor components Co' C4, and Cs contain
`ClO, C12, and C12 fatty acids (1). The ratios offactors Cl' Cz' and C3' which are nor(cid:173)
`mallyabout 1:1.5:1, can be modulated by adding different branched-chain amino acids to
`the fermentation medium. Factor Cz can be increased by adding valine to the fermenta(cid:173)
`tion, whereas factors C 1 and C3 can be increased by adding isoleucine (7). The authors
`concluded that the branched-chain amino acids served as precursors to provide the
`branched-chain fatty acid primers for the biosynthesis of the fatty acid side chains of the
`A21978C factors.
`Daptomycin, which contains the ClO fatty acid decanoic acid, is normally pro(cid:173)
`duced by S. roseosporus in trace amounts. Huber et al. (8) have shown that decanoic acid
`mixed 1: 1 (v:v) in methyl oleate can be fed continuously to fermenters at rates that avoid
`the accumulation of decanoic acid, which is normally toxic to S. roseosporus. Under
`these conditions, S. roseosporus A21978.65 produced over 900 )lg/ml of daptomycin
`directly, and less than 400 )lg/ml of factors containing other fatty acid side chains. The
`process was modified for large-scale production, and daptomycin yields of > 1000 )lg/ml
`representing 77% of total A21978C factors have been reported (9). High yields of dapto(cid:173)
`mycin can also be produced by feeding caproic acid (10). This process is much simpler
`and less costly than the process that combines enzymatic removal of the fatty acid side
`chains and chemical reacylation with decanoic acid (see Section III).
`
`B. AS414S Biosynthesis
`The cyclic lipopeptide antibiotic complex A54145 is produced by S. fradiae A54145
`(NRRL 18158). The complex has eight factors composed of four different cyclic pep tides
`
`PETITIONERS
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`EXHIBIT NO. 1008 Page 4 of 23
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`

`

`I
`t '
`\
`
`L-Asp
`
`L-Om
`
`D-Ser
`\
`
`(L-threo)3-MeGlu
`
`0
`
`/L-Asp ..........
`D-Ala
`GIY\
`
`LIPOPEPTIDE ANTIBIOTICS 417
`
`I
`L-Kyn
`,<
`'L-t ....... O
`L-rp
`L-1sn
`L-Trp
`k
`~o
`
`Gly
`
`(a)
`
`Daptomycin - LY146032
`
`Figure I Structures of daptomycin and A54145B,. Daptomycin (a) contains an N-decanoyl side
`chain. The A21978C factors produced in standard fermentations contain the fatty acyl side chains
`shown in Table 1. A54145B, (b) is the most abundant factor produced in standard fermentations.
`The other A54145 factors are shown in Table 2.
`
`Table I Upopeptide A21978C Factors Produced
`by S. roseosporus
`
`Factor
`
`Fatty acid
`
`Unidentified (10 carbon)
`anteiso-Undecanoyl
`iso-Dodecanoyl
`anteiso-Tridecanoyl
`Unidentified (12 carbon)
`Unidentified (12 carbon)
`
`Data from Ref. 1.
`
`and three different lipid side chains (4,5). The major peptide nucleus has the sequence
`Trp-Glu-hAsn-Thr-Sar-Ala-Asp-Lys-OmAsp-Gly-Asp-3mGlu-Ile (where hAsn =
`hydroxy Asn, OmAsp = hydroxy methyl Asp, and 3mGlu = 3-methyl Glu). The peptide
`is cyclized by an ester linkage between the carboxy-group of He and the Thr hydroxy(cid:173)
`group. The four different peptide structures are identical in the first 11 amino acids, but
`have 3mGlu or Glu at position 12 and He or Val at position 13. The different peptide
`structures coupled with three different possible lipid side chains account for the eight fac(cid:173)
`tors identified in fermentation broths (Table 2). (Presumably, the four missing factors
`were present in such low amounts that they were not observed by the high-performance
`liquid chromatography [HPLC] system used.)
`
`PETITIONERS
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`EXHIBIT NO. 1008 Page 5 of 23
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`

`418 Baltz
`
`Table 2 Lipopeptide A54145 Factors Produced by
`S.' fradiae NRRL18158
`
`Amino acid at position:
`
`Factor
`
`12
`
`A
`Al
`B
`BI
`C
`0
`E
`F
`
`Glu
`Glu
`3mGlu
`3mGlu
`3mGlu
`Glu
`3mGlu
`Glu
`
`Data from Ref. 4.
`
`13
`
`lie
`lie
`lie
`lie
`Val
`lie
`lie
`Val
`
`Fatty acid
`
`8-Methylnonanoyl
`n-Decanoyl
`n-Decanoyl
`8-Methylnonanoyl
`8-Methyldecanoyl
`8-Methyldecanoyl
`8-Methyldecanoyl
`8-Methyldecanoyl
`
`S. fradiae NRRL 18158 produces> 1 mg/m of A54145 factors in a glucose-fed com(cid:173)
`plex medium (4). Factors A and Bl' which differed from each other at position 12, were
`the most abundant factors produced. Factor A, containing Glu at position 12, was syn(cid:173)
`thesized more rapidly than Bl early in the fermentation, then stopped accumulating at
`about 135 hr. Factor Bl' containing 3mGlu, continued to be synthesized up to 180 hr,
`and it was the most abundant factor at the end of the fermentation. Since A54145 is syn(cid:173)
`thesized by a thiotemplate mechanism by a large multisubunit peptide synthetase (see
`below), the accumulation of factors A and B1 suggests several possibilities. One is that
`early accumulation of factor A and late accumulation of factor B1 are 'due to a precur(cid:173)
`sor-product relationship between factor A and factor Bl' and that the methylation of
`Glu occurs after the peptide is synthesized. A second possibility is that Glu is converted
`to 3mGlu in the cytoplasm, and that the peptide synthetase can accept either amino acid
`for incorporation. The change in rates of biosynthesis of factors A and Bl would reflect
`the relative availability of Glu and 3mGlu during the course of the fermentation-the
`latter predominating late in the fermentation. A third possible explanation is that methy(cid:173)
`lation of Glu occurs after binding to the peptide synthetase. In this case, the relative
`amounts of factors A and B1 could be influenced by the level of methyl donor, presum(cid:173)
`ably S-adenosyl methionine, during the fermentation. Late accumulation of factor Bl
`would reflect a higher level of methyl donor late in the fermentation. The cloning and
`sequence analysis of genes involved in A54145 biosynthesis, coupled with gene disrup(cid:173)
`tion analysis, should shed light on this question.
`The substitution of Val for He at position 13 is a simpler problem. In this case, fac(cid:173)
`tors containing Val are normally produced in very low quantities, and they are not
`detectable until 90 hr in the fermentation (4). However, the ratios of the different
`A54145 factors containing He or Val can be altered by feeding the amino acid precursors
`(11). The feeding of Val had two effects. It caused an increase in factors containing Val
`at position 13 from 10 to 54%, and it caused the accumulation of nearly 100% of factors
`containing 8-methylnonanoyl side chains. The feeding of He caused a reduction in fac(cid:173)
`tors containing Val at position 13 from 10 to 0%, and it caused an increase in factors
`containing the 8-methyldecanoyl side chain. These results indicate that the peptide syn(cid:173)
`thetase module that catalyzes the binding and addition of amino acid for position 13 has
`
`PETITIONERS
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`EXHIBIT NO. 1008 Page 6 of 23
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`

`LIPOPEPTIDE ANTIBIOTICS 419
`
`relatively loose substrate specificity, and can bind, activate, and couple Valor He to the
`growing chain. Furthermore, the enzyme that carries out the formation of the ester bond
`with Thr at position 4 can tolerate Valor He as substrates in the ring closure reaction.
`The·distribution of fatty acid side chains can also be modulated by feeding different fatty
`acids. Continuous feeding of ethyl caprate increased the factors containing the n(cid:173)
`decanoyl si'de chain from 13 to 82% at the expense of factors containing 8-methyl(cid:173)
`nonanoyl or 8-methyldecanoyl side chains. The addition of methyl oleate, decyl alde(cid:173)
`hyde, or decyl alcohol gave a similar response. The feeding of other shorter-chain fatty
`acids led to the production of novel derivatives containing shorter side chains. For exam(cid:173)
`ple, feeding of hexanoic acid resulted in 96% of factors containing C6, and feeding
`nonanoic acid gave 100% of factors containing C9. Therefore, the enzymatic step
`involved in the coupling of the fatty acid to the amino-group ofTrp is indiscriminant in
`its specificity for fatty acids with chain lengths of C6 to Cll.
`
`C. Peptide Synthetase Structure and Function
`Like many other linear and cyclic peptide secondary metabolites, daptomycin and
`A54145 are produced by a nonribosomal thiotemplate mechanism. The peptide syn(cid:173)
`thetases involved in these processes are generally very large, being composed of one or
`more multienzyme subunits (12-19), and contain about 125 kDa per amino acid incor(cid:173)
`porated. By analogy, the peptide synthetases for daptomycin and A54145 should be about
`1.6 mDa in size. Wessels et al. (20) have been studying the peptide synthetases involved
`in daptomycin and A54145 biosynthesis. They identified high-molecular-weight proteins
`from transition phase or early stationary phase cultures of S. roseosporus and S. fradiae
`that reacted to antibodies prepared against the Streptomyces chrysomallus actinomycin
`synthetase 2 or to antibodies prepared against the SGTTG sequence conserved in adeny(cid:173)
`late-forming enzymes. Their data suggest that the daptomycin and A54145 synthetases
`have similar structures. Each multi enzyme appears to have three subunits, two of about
`700 kDa and one about 250 kDa. The 250 kDa subunit of the daptomycin synthetase was
`purified and shown to activate L-Kyn. This suggests that the 250 kDa subunits contain
`the two domains responsible for the addition of the final two amino acids. A number of
`other amino acids were activated by the larger subunits, and the data indicated that only
`L-amino acids were activated, so that epimerizations must occur on the peptide syn(cid:173)
`thetase as observed with other peptide synthetases. Also, N-methylation of Gly must
`occur on the enzyme, since Gly but not sarcosine (Sar) was activated by the A54145 syn(cid:173)
`thetase. Further work is needed to define what domains are associated with the individual
`high-molecular-weight subunits. Gene disruption studies may be particularly useful, since
`they would eliminate the need to purify the individual subunits to homogeneity.
`
`III. ENZYMATIC AND CHEMICAL MODIFICATION OF LlPOPEPTIDES
`
`To study the structure-activity relationships of the fatty acyl side chains, it was desirable
`to have a method to remove the natural fatty acids from the A21978C complex of antibi(cid:173)
`otics. The fatty acid side chains could not be removed chemically without significant
`destruction to the peptide core. However, the side chains were readily removed by incu(cid:173)
`bation of the complex with a culture of A. utahensis (2,3). Enzymatic de acylation resulted
`in complete loss in antibacterial activity (2). Deacylation also occurred with A2l978C
`
`PETITIONERS
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`EXHIBIT NO. 1008 Page 7 of 23
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`420 Baltz
`
`complex containing tert-butoxycarbonyl (t-BOC)-protected amino function of the side
`chain of Om. Deacylation occurred most efficiently with A. utahensis cultures grown for
`65 to 85 hr, and conversion efficiencies of ~85% were achieved in 2 hr incubation at pH
`7.0-8.0. Conversion rates of >600 Jlg/ml per hr were achieved. Deacylase activity was
`also observed with three other Actinoplanes strains and a strain of Streptosporangium
`roseum.
`The ability to block the amino function of Om, then deacylate the t-BOC-pro(cid:173)
`tected nucleus, has facilitated the development of many A21978C anaiogs containing
`modifications at the N-terminal Trp. Semisynthetic derivatives containing novel fatty
`acyl, aroyl, and extended peptide side chains were readily deb locked by trifluoroacetic
`acid treatment (3). Subsequent biological studies identified the n-decanoyl derivative,
`daptomycin, as a candidate for clinical development (see Section IV).
`Similar studies have shown that the £N-BOC-L-Lys-protected A54145 complex
`can be deacylated by A. utahensis, the N-terminus of Trp reacylated with different fatty
`acids, and the protected Lys deblocked to give novel semisynthetic antibiotics for in vitro
`and in vivo testing (6). Several of these derivatives are discussed in Section IV.
`
`IV. ANTIBACTERIAL ACTIVITY OF DAPTOMYCIN, AS414S, AND
`RELATED LlPOPEPTIDES
`In Vitro Studies
`A.
`Table 3 summarizes the in vitro antibacterial activity of daptomycin against Gram-posi(cid:173)
`tive bacteria. Daptomycin is very active against most Gram-positive pathogens, including
`strict anaerobes, enterococci, staphylococci, and streptococci. Daptomycin is active
`against antibiotic-resistant pathogens, including methicillin-resistant S. aureus and S.
`epidermidis, penicillin-resistant S. pneumoniae, and vancomycin-resistant Enterococcus
`faecalis and Enterococcus faecium (Table 4). This reflects its novel mechanism of action,
`which differs from that of p-Iactam and glycopeptide antibiotics (see Section V). Dapto(cid:173)
`mycin is bactericidal in Gram-positive pathogens, including enterococci (26,36-38), and
`the rate of killing is dose-dependent (39-43). Antibacterial activity of daptomycin is cal(cid:173)
`cium-dependent, and the presence of serum or albumin decreases the bactericidal activ(cid:173)
`ity of daptomycin (36,44-46). Daptomycin is >90% bound to albumin in serum or in
`media containing physiological concentrations of albumin (46-48). This work will be
`discussed in Section IV.C in relation to daptomycin clinical trials. Daptomycin shows
`synergy with streptomycin (38), gentamicin (49), and tobramycin (49) in its bactericidal
`activity against enterococci. It also shows synergy with imipenen, amikacin, netilmicin,
`and fosfomycin in its bactericidal effects on methicillin-sensitive and -resistant strains of
`S. aureus and S. epidermidis (50). Interestingly, daptomycin seems to protect experimental
`animals against tobramycin and gentamicin renal toxicity (51-54).
`_ The in vitro antibacterial activities of the A54145 factors are similar to those of
`the A21978C factors, and minimal inhibitory concentrations (MICs) ranged from 0.5 to
`32 Jlg/ml against various strains of S. aureus, S. epidermidis, Streptococcus pyogenes, and
`enterococci (55). Table 5 shows in vitro activities of a series of A54145A derivatives
`containing different fatty acid side chains compared with those of a comparable series of
`A21978C derivatives, including daptomycin. The A21978C series were generally 2- to
`16-fold more active when comparisons are made between compounds containing identi(cid:173)
`cal fatty acid side chains. However, A54145A containing C14 chain length was at least
`
`PETITIONERS
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`EXHIBIT NO. 1008 Page 8 of 23
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`

`

`Table 3
`
`In Vitro Antibacterial Activity of Daptomycin Against Gram-Positive Bacteria
`
`LIPOPEPTIDE ANTIBIOTICS 421
`
`Microorganism (no. of strains)
`
`Actinomyces spp. (12)
`Bifidobacterjum spp. (10)
`Clostridium difficile (170)
`Clostridium perfringens (40)
`Corynebacterium jeikeium (18)
`Enterococcus faecaUs (199)
`Enterococcus faecium (10)
`Eubacterium aerofaciens (10)
`Eubacterium lentum (10)
`Lactobacillus acidophilus (13)
`Lactobacillus casei (18)
`Lactobacillus plantarum (18)
`Lactococcus spp. (32)
`Leuconostoc spp. (13)
`Listeria monocytogenes (57)
`Pediococcus (6)
`Peptostreptococcus anaerobius (16)
`Peptostreptococcus asaccharolyticus (32)
`Peptostreptococcus magnus (11)
`Peptostreptococcus micros (12)
`Peptostreptococcus prevotii (9)
`Peptostreptococcus productus (9)
`Propionibacterium acnes (43)
`Staphylococcus aureus (436)
`Staphylococcus epidermidis (335)
`Staphylococcus haemolyticus (50)
`Staphylococcus hominis (20)
`Staphylococcus saprophyticus (20)
`Streptococcus agalactiae (37)
`Streptococcus avium (10)
`Streptococcus bovis (10)
`Streptococcus lactis (2)
`Streptococcus pneumoniae (91)
`Streptococcus pyogenes (60)
`Streptococcus sangius (7)
`
`MIC50 (Ilg/ml)
`
`References
`
`0.25
`0.5
`0.12
`0.25,4.0
`0.12
`0.5-2.0
`4.0
`1.0
`4.0
`2.0
`1.0
`1.0
`0.5
`0.5
`1.0-8.0
`0.5-1.0
`0.12
`0.12
`0.12
`0.12
`0.12
`0.12
`0.5,4.0
`0.25-0.5
`0.25-0.5
`0.25
`0.25
`0.25
`0.13-0.5
`1.0
`0.25
`0.25
`0.06-0.5
`0.06--0.25
`0.5
`
`21
`21
`21-23
`21,24
`24
`23-27
`25
`21
`21
`21
`21
`21
`28
`28
`24, 25, 29-31
`28
`21
`21
`21
`21
`21
`21
`21, 24
`23-25,29,30,32
`23-25,29,30,32
`30-32
`32
`32
`24-26,32
`25
`24
`21
`24-26,29,30
`24,26,32
`31
`
`as active as daptomycin. The A54I45A derivatives have not been studied extensively for
`their activity against an extended set of Gram-positive bacteria because of their disap(cid:173)
`pointing in vivo activities, discussed below.
`
`In Vivo Studies
`B.
`Many derivatives of A2I978C have been examined for their in vivo activities against S.
`aureus and S. pyogenes using mouse protection tests (3). Generally, derivatives containing
`relatively short-chain fatty acid side chains (C6-C9) were less active than daptomycin
`(CI0) and derivatives containing Cll-CI4. However, compounds containing Cll-CI4
`were progressively more toxic to mice as the side chain length increas~d (see Table 6 for
`
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`

`422 Baltz
`
`Table 4
`
`In Vitro Antibacterial Activity of Daptomycin Against Antibiotic-Resistant Pathogens
`
`Microorganism (no. of strains)
`
`Enterococcus faecalis (2)
`I
`Enterococcus faecalis (1) BM4166
`Enterococcus faecalis (1)
`Enterococcus faecalis (1)
`Enterococcus faecium (1)
`Enterococcus faecium (1)
`Enterococcus faecium (1)
`Enterococcus faecium (1)
`Staphylococcus aureus (240)
`Staphylococcus aureus (261)
`Staphylococcus epidermidis (189)
`Staphylococcus epidermidis (176)
`Staphylococcus haemolyticus (60)
`Staphylococcus haemolyticus (85)
`Streptococcus pneumoniae (10)
`
`Antibiotic
`resistancea
`
`Vans ErmS
`VanR ErmR
`Vans Erms
`VanR ErmR
`Vans Erms
`Vans ErmR
`VanR Erms
`VanR ErmR
`Mcns
`McnR
`Mcns
`McnR
`OcnS
`OcnR
`PenR
`
`MIC or MICsob
`
`References
`
`2
`2
`0.5
`0.5
`2
`2
`2
`2
`0.12--0.5
`0.25--0.5
`0.12--0.25
`0.25
`0.25,0.5
`0.25,0.5
`0.5
`
`33
`33
`34
`34
`33
`33
`33
`33
`24-27,29,32,35
`24-27,29,32,35
`23,24,26,27,29,32,35
`23,24,26,27,29,32,35
`31,32
`31,32
`25
`
`aVan, vancomycin; Erm, erythromycin; Men, Methycillin; Oen, oxacillin; Pen, penicillin;S, sensitive;R, resis(cid:173)
`tant.
`b MIC is given for single isolates; MICso is given for multiple isolates.
`
`Table S
`
`In Vitro Activities of Related Lipopeptide Antibiotics Against Gram-Positive Pathogens
`
`MIC (/lg/ml)
`
`Cyclic peptide nucleus
`
`Lipid side chain
`
`S. aureus
`
`S. epidermidis
`
`E. faecalis
`
`A21978C
`A21978C
`A21978C
`A21978C
`A21978C
`A54145A
`A54145A
`A54145A
`A54154A
`A54145A
`
`Data from Refs. 3 and 55.
`
`Nonanoyl
`Decanoyl
`Undecanoyl
`Dodecanoyl
`Tetradecanoyl
`Nonanoyl
`Decanoyl
`Undecanoyl
`Dodecanoyl
`Tetradecanoyl
`
`8
`0.5
`0.25
`0.5
`0.5
`32
`8
`2
`1
`0.25
`
`8
`0.5
`0.5
`0.5
`2
`16
`4
`2
`1
`0.5
`
`128
`16
`2
`4
`0.5
`128
`64
`16
`4
`2
`
`typical results}. Calculation of the relative therapeutic index (mouse lethal dose/effica(cid:173)
`cious dose [LDsolEDso]) suggests that the CIO derivative (daptomycin) is superior to the
`others. By comparison, the A54145 derivatives were generally much less active in the
`mouse EDso tests, but they showed similar levels of toxicity in the mouse LDso tests.
`Thus, the relative therapeutic indexes of the A54145 derivatives are roughly IOO-fold
`lower than that of daptomycin. For example, the dodecanoyl derivative of A54145A,
`which was only 2-fold less active than daptomycin in vitro against S. aureus and S. epi-
`
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`LIPOPEPTIDE ANTIBIOTICS 423
`
`Table 6 Comparison of In Vivo Activities of Selected Lipopeptide
`Antibiotics Against S. pyogenes
`
`Compound
`
`Mouse ED50 Mouse LD50
`
`Relative therapeutic
`index (LD501ED50)
`
`i\21978C,
`A21978Cz
`A21978C3
`A21978C heptanoyl
`A21978C octanoyl
`A21978C nonanoyl
`A21978C decanoyl
`A21978C undecanoyl
`A21978C dodecanoyl
`A54145A nonanoyl
`A54145A decanoyl
`A54145A undecanoyl
`A54145A dodecanoyl
`A54145F decanoyl
`A54145B decanoyl
`
`0.064
`0.03
`0.032
`1.49
`0.65
`0.14
`0.03
`>0.06
`0.05
`10.06
`5.0
`1.6
`1.2
`10.08
`0.94
`
`>600
`175
`75
`>600
`>600
`>600
`600
`450
`144
`>500
`>500
`>500
`321
`>500
`28
`
`>9,375
`5,833
` 2,344
`>402
`>923
`>4,286
`20,000
`<7,500
`2,880
`>47
`>100
`>313
`268
`>46
`30
`
`Data from Refs. 3 and 55. See Table 1 for A21978C p C 2, and C3 structures.
`
`dermidis and 4-fold more active against E. faecalis, (Table 5) had a relative therapeutic
`index against S. pyogenes 75-fold lower than that of daptomycin (Table 6). The data sug(cid:173)
`gest that the mouse toxicity is determined primarily by the fatty acid side chain length,
`and the in vivo antibacterial activity is determined by a combination of the amino acid
`sequence of the depsipeptide and the fatty acid side chain length.
`Daptomycin-treated Clostridium difficile colitis was examined in a hamster model
`(56). Daptomycin was equal in in vitro activity to vancomycin, but it was 100 times as
`active as vancomyin in vivo in this model. The authors suggested that the difference in
`in vivo efficacy may be due to the bactericidal activity of daptomycin. Daptomycin has
`also been shown to treat S. aureus endocarditis in a rabbit model, and these studies will
`be discussed in Section IY.D in the context of the human clinical studies.
`
`C. Clinical Studies
`Daptomycin was shown to be well tolerated in normal human volunteers when given
`intraveniously in a 30 or 60 min infusion at 1 or 2 mg/kg every 24 hr (41,57). Wood(cid:173)
`worth et al. (58) also studied the safety, pharmacokinetics, and disposition of daptomycin
`in healthy volunteers. They showed that in single intravenous doses infused over 30 min,
`daptomycin was well tolerated at 0.5 to 6.0 mg/kg per day. An average of 96,4% of dap(cid:173)
`tomycin was bound to protein in the serum. About 78% of radiolabeled daptomycin was
`recovered in the urine, but about one-third of the radioisotope was present in uncharac(cid:173)
`terized metabolites lacking antibacterial activity. The relatively long serum half life (Tl/z)
`of ~8 hr and the high serum levels of daptomycin achieved at doses of 4 or 6 mg/kg indi(cid:173)
`cated that adequate antibacterial levels can be achieved at 95% serum binding. Direct
`tests of serum samples for antibacterial activity against S. aureus and E. faecalis confirmed
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`424 Baltz
`
`this. The authors cautioned that the high protein binding and large molecular size of
`daptomycin may limit the distribution of daptomycin out of the plasma, and so dapto(cid:173)
`mycin treatment of deep-seated infections, such as bone infections and endocarditis, may
`have limited-effectiveness.
`At a dose of 2 mg/kg every 24 hr, daptomycin was shown to be effective in treating
`a variety of Gram-positive infections (59). In another study, daptomycin given at a dose
`of 3 mg/kg every 12 hr was shown to be effective in treating Gram-positive bacteremias
`and endocarditis caused by Gram-positive pathogens (including E. faecalis) other than S.
`aureus (60). Only two of seven patients with S. aureus endocarditis had successful out(cid:173)
`comes. Five patients were discontinued from the study because of adverse effects. In
`another study, daptomycin at 2 mg/kg once a day failed to treat two very seriously ill
`patients with Gram-positive infections (61). It was suggested in ,the latter two studies
`that higher doses of daptomycin would be required to treat S. aureus endocarditis and
`severely ill patients. However, the occasional adverse effects noted at a dose of 3 mg/kg
`every 12 hr seemed to preclude raising the dose further, and clinical trials were stopped.
`Rybak et al. (46), during the clinical evaluation of a higher-dose regimen of dapto(cid:173)
`mycin, observed that within a group of intravenous drug abusers, daptomycin peak serum
`concentrations were lower and volumes of distribution were higher than those reported
`in healthy volunteers. They also noted that serum albumin levels tended to be low in this
`group. They noted that patient-specific variability that may lead to lower than expected
`peak daptomycin concentrations, as exemplified by intravenous drug abusers, coupled
`with high serum binding, may impair the efficacy of the drug, and this may have con(cid:173)
`tributed to some failures in clinical trials. They also showed that the bactericidal activity
`of daptomycin is greater in exponentially growing S. aureus cultures than in stationary
`phase cultures, and that this may be an additional factor in the poor efficacy of dapto(cid:173)
`mycin in treating S. aureus endocarditis.
`Lee et al. (62) studied serum binding in six human volunteers given an intravenous
`infusion of daptomycin at 3 mg/kg, and demonstrated that the average binding was 90%.
`They noted that the total serum concentration of daptomycin was about 30 Jlg/ml30 min
`after infusion. However, the unbound daptomycin dropped to 0.37 Jlg/ml by 12 hr. This
`was below the MIC for enterococci and close to the MIC for staphylococci. They sug(cid:173)
`gested that the serum binding may have been part of the reason that clinical failures were
`observed in the once-a-day protocol using 2 mg/kg, and that higher daptomycin concen(cid:173)
`trations at more frequent dosing intervals would be needed to maintain adequate unbound
`concentrations of daptomycin.
`
`D. Postclinical Studies
`Hanberger et al. (45) showed that in the presence of physiological concentrations of
`albumin and Ca2+, the MICs and postantibiotic effects (PAEs) of daptomycin increased
`about two-fold against S. aureus and E. faecalis. This effect appears to be due to the bind(cid:173)
`ing of daptomycin to albumin, which has been shown to be >90% in other studies. How(cid:173)
`ever, the PAEs at clinically achievable concentrations were still >6 hr. The authors sug(cid:173)
`gested that the low dose concentrations achieved in the suspended clinical trials of
`daptomycin may not have been sufficient to eradicate the susceptible infecting organ(cid:173)
`isms, but the failure may also have been due to insufficient Ca2+ concentrations at the
`infectious foci, thus reducing daptomycin antibacterial activity.
`Garrison et al. (44) set up an in vitro model to assess the effects of serum albumin
`on the killing rate of an S. aureus strain isolated from a patient unsuccessfully treated by
`
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`LIPOPEPTIDE ANTIBIOTICS 425
`
`daptomycin in a clinical trial. High and low dose regimens were tested in the model.
`Th~y showed that the killing rate was reduced 3-fold in the presence of albumin in the
`high-dose daptomycin simulation and 10-fold in the low-dose simulation. They con(cid:173)
`cluded that the presence of albumin can dramatically decrease the activity of daptomycin
`against S. auri'!US, and that the magnitude of the loss is related to daptomycin concentra(cid:173)
`tion. They al~o concluded that larger daily doses of daptomycin than those used in the
`early clinical trials of daptomycin (2 mg/kg per day; see Section IV.C) are more likely to
`be successful in the management of bacteremia and endocarditis in humans.
`In an attempt to understand the treatment failures in the phase II clinical trial of
`daptomycin, Lamp et al. (47) carried out studies to explore the effects of inoculum size,
`growth phase, and pH on the bactericidal activity of daptomycin in serum. They noted
`that S. aureus endocarditis represents an infection characterized by a high bacterial inocu(cid:173)
`lum, impaired host defense, and low metabolic activity in a nutritionally deficient envi(cid:173)
`ronment. They showed that the MICs